JP2019503341A - Targeted delivery of therapeutic proteins biologically encapsulated in plant cells to cell types of interest for the treatment of disease - Google Patents

Abstract

Disclosed are compositions and methods for producing a therapeutic fusion protein comprising a target sequence that directs the protein to a cell or tissue of interest.

Description

This application claims priority to US Provisional Application No. 62 / 256,053, filed Nov. 16, 2015, the entire disclosure of which is fully incorporated herein by reference. And
The US government has the rights to the present invention using funds from National Institutes of Health grant numbers R01 GM63879, R01 HL 109442, and R01 HL 107904.

The present invention relates to the field of transplastomic plants (plants with genetically engineered chloroplasts) and the production and supply of low cost protein pharmaceuticals. More specifically, the present invention provides a transgene encoding a therapeutic protein and peptide operably linked to a target sequence that directs plant cells encapsulating the fusion protein to a tissue of interest upon treatment in the intestine. Plants comprising chloroplasts that express genes (transgenes) are provided.

BACKGROUND OF THE INVENTION In order to illustrate the state of the art to which this invention pertains, several publications and patent documents are cited throughout the specification. Each of these citations is hereby incorporated by reference as if fully set forth.

  Biopharmaceuticals produced with current systems are very expensive and not affordable for the majority of the world population. In the United States, the average annual cost of protein drugs is 25 times higher than that of small molecule drugs. The cost of protein drugs ($ 140 billion in 2013) exceeds the GDP of more than 75% of countries around the world and is unreachable in these countries [1]. One third of the world's population with an income of less than $ 2 per day cannot purchase any protein drugs. Recombinant insulin has been sold commercially for 50 years, but is still not affordable for the majority of the world population. This is because the production of such drugs is prohibitively expensive and often requires expensive fermenters, multi-step purification procedures, refrigeration / transport, and sterile delivery means. Furthermore, the short shelf life of these drugs is associated with increased costs. Oral delivery of protein drugs has not received much attention for decades because they cannot cross the intestinal epithelium because of their degradation in the digestive system and delivery to target cells.

However, some recent studies have indirectly shown that plant cell walls protect expressed protein drugs from gastric acids and enzymes through biological encapsulation [2, 3]. . Human digestive enzymes are unable to break down the glycosidic bonds of carbohydrates that make up the cell walls of plants. However, when natural plant cells containing a protein drug reach the intestine, commensal microorganisms digest the plant cell wall and release the protein drug in the intestinal lumen. Bacteria that inhabit the human intestine have evolved to utilize complex carbohydrates in the plant cell wall and can utilize almost all plant glycans [4,5].
A fusion of (non-toxic) cholera toxin B subunit (CTB) to green fluorescent protein (GFP) expressed in chloroplasts and bioencapsulated in plant cells via the GM1 receptor Delivered through the intestinal epithelium and GFP was released into the circulatory system [6]. Fusion of CTB to therapeutic protein led to oral tolerance [7-11] or induction of functional proteins into serum [12-14], and even blood brain or retinal barriers, Facilitates effective oral delivery [15, 16].

  Foreign proteins can also be delivered to living cells by fusion with protein transduction domains (PTDs) with cell membrane permeation properties that do not require specific receptors [17]. Peptide and protein transduction domains (PTDs) are small cationic peptides containing 8 to 16 amino acids and most often function as transporters for the delivery of macromolecules [17 ].

  PTD carries molecules into cells by receptor-independent liquid-phase macro-pinocytosis, a special form of endocytosis. Different PTDs show similar cellular uptake properties, but they differ in the efficiency of transporting protein molecules into the cell.

  It has been found that the effectiveness of cellular uptake correlates strongly with the number of basic amino acid residues. Since PTD has been shown to deliver biologically active proteins in cultured mammalian cells and in vivo and in vitro animal models [18-20], methods for delivering PTD fusion proteins include: Has the potential to deliver large therapeutic drugs.

T lymphocytes and B lymphocytes are major cellular components of the adaptive immune response, but their activation and homeostasis are controlled by dendritic cells. B cells can recognize natural Ag and secrete antibodies directly through B cell receptors on their surface. However, T cells can only recognize peptides displayed by MHC class I and II molecules on the surface of APC.
Macrophages are a type of “professional” antigen-presenting cells that have many important roles, including the removal of dead cells and cell debris and the initiation of immune responses in chronic inflammation [ 21, 22].

  Macrophages are involved in orchestration of primary and secondary immune responses. Mast cells are involved in generating an initial inflammatory response during infection, which is important for initiating innate and adaptive immunity. When activated, mast cells rapidly release their unique granules and various hormone mediators into the interstitium. Thus, mast cells play an important role in wound healing, allergic diseases, anaphylaxis and autoimmunity.

Dendritic cells are important immunoregulatory cells. Dendritic cells form a complex with multifunctional APC and play an important role in anti-pathogen activity. Furthermore, dendritic cells differentiate into different types of functional cells, are stimulated by different antigens, and induce humoral or cellular immunity.
Conversely, DC is also important for regulatory T cell (Treg) homeostasis, Treg extrathymic induction, induction of immune tolerance in transplantation, and treatment of allergy or autoimmune diseases It is.

Tissue microenvironment, activation signals, and subsets of DC are important parameters that determine whether antigen presentation by DC results in immunity or resistance [23-25].
Thus, targeted in vivo delivery of antigens to DCs is not only useful for inducing tumor-specific immune responses and establishing new strategies for vaccine development, cancer immunotherapy, but also tolerance induction protocols (tolerance) (instruction protocols) [26-28].
For example, gut-associated lymphoid tissue (GALT) is a tolerogenic property that includes maximum surface area for entry of antigens into the body and expression of the immunosuppressive cytokines IL-10 and TGF-b. Provides a very unique microenvironment with [29-31].

Sample antigens of intestinal epithelial cells and CX1CR5 ⁺ macrophages from the intestinal lumen. In particular, in Peyer's patch endothelium, microfold cells (M cells) take up and phagocytose antigens, leading them to DC.
Intestinal CD11c ⁺ DC preferentially contains a high proportion of CD103 ⁺ DC expressing TGF-β that induces Treg cells [32].

Recently, upon delivery of bioencapsulated CTB fusion antigen, we have increased oral tolerance induction against coagulation factors in hemophilic mice with increased CD103 ⁺ DC We demonstrated that it was associated with frequency, antigen uptake by CD103 ⁺ DC, and induction of several subsets of Tregs [9, 10].

  There was also an increase in plasmacytoid DCs with important immunoregulatory functions [33]. DC peptide (DCpep) was developed as a ligand to mucosal DC [26]. This small peptide binds to a DC-specific receptor and facilitates transport of macromolecules to DC.

SUMMARY OF THE INVENTION In accordance with the present invention, a method for targeted delivery of a therapeutic protein to a tissue or cell of interest in a subject in need thereof is disclosed. An exemplary method comprises administering an effective amount of a plant cell or residue thereof comprising a plastid-expressing nucleic acid encoding said therapeutic protein operably linked to a fusion peptide sequence;
Expression of the nucleic acid causes the production of a therapeutic fusion protein that is selectively delivered in vivo to the target cell or tissue of interest upon ingestion or ingestion of the plant or its remnants, To selectively deliver the therapeutic protein to the target cell or tissue of interest. Preferably, the plant is a green leafy vegetable including but not limited to lettuce, low nicotine tobacco, spinach, cabbage and kale. Other plants include eggplant, carrot, and tomato.

In one embodiment, the fusion peptide sequence is PTD. In another embodiment, the fusion peptide sequence is a DC peptide. The fusion peptide may be an antimicrobial peptide such as PG1 or RC101. In certain embodiments, the fusion peptide is a CTB peptide.
In other embodiments of the invention, the use of CTB fusion peptides is ruled out, particularly when targeting needs to be more specific.
Targeted cells include, but are not limited to immune cells, somatic cells and dendritic cells. Tables 2, 3 and 4 provide useful therapeutic molecules for the plastid producing fusion proteins of the invention.

In one aspect, the therapeutic fusion protein is for the treatment of an endocrine disorder, and the protein is selected from the group consisting of insulin, somatotropin, and insulin-like growth factor.
The therapeutic fusion protein can also be used for maintaining hemostasis or preventing thrombus.
In this embodiment, the therapeutic fusion protein is selected from the group consisting of blood clotting factor, antithrombin, protein C and tissue plasminogen activator.
The methods of the invention can also be used to treat subjects with enzyme deficiencies, and therapeutic fusion proteins include β-gluco-cerebrosidase, α-glucosidase, lactase, lipase, amylase, protease, and proteinase. A protein selected from the group consisting of inhibitors.

  The therapeutic fusion proteins can augment existing biological pathways, including erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, follicle stimulating hormone, chorionic gonadotropin, alpha interferon, interferon B, PDGF, Selected from the group consisting of keratinocyte growth factor and bone morphogenetic protein.

Another aspect of the present invention includes a plant or plant cell comprising the therapeutic fusion protein. The plant may be lyophilized. These may be in a powder form or optionally encapsulated.
In preferred embodiments, the therapeutic fusion protein is stable for several months at ambient temperature.

1A-1F.
Schematic diagram of predicted protein structure and properties of transplastomic lines expressing GFP fusion protein
Interaction of CTB fusion protein with GM1 receptor and predicted 3D structure of both PTD and DCpep. The pentasaccharide portion of the GM1 receptor establishes an interaction with the pentameric structure of CTB. A hinge sequence to avoid steric hindrance and a furin cleavage site to release the tethered protein was placed between the CTB and the fusion protein. The computationally predicted three-dimensional structures of both PTD and DCpep were obtained from an I-TASSER (iterative threading assembly refinement) server [40]. This structure is shown in iridescent color and gradually changes from blue to red (blue-green-yellow-orange-red) from N-terminal to C-terminal residues. Among the predicted structures, a confidence score (C-score), a high-resolution model with root mean square deviation (RMSD) value (high-resolution models with root mean square), and a template modeling score ( A model with the most reliable structure for each peptide, selected based on combined results from parameter calculations such as template modeling score (TM-score), was presented. Schematic of GFP fusion carrier protein expression cassette and flanking regions. Prrn: rRNA operon promoter; aadA: aminoglycoside 3'-adenylyltransferase gene; PpsbA: 5'UTR of promoter and psbA gene; CTB: coding sequence of non-toxic cholera B subunit; PTD: coding sequence of protein transduction domain; DCpep: dendritic cell binding peptide sequence; smGFP: gene sequence of soluble modified green fluorescent protein; TpsbA: 3'UTR of psbA gene; trnI: isoleucyl-tRNA; trnA: alanyl-tRNA. Restriction enzymes used for Southern blot analysis were indicated as BamHI / BglII for probe generation and HindIII for digestion of genomic DNA. Southern blot analysis of each transplastomic line expressing a GFP fusion tag protein. HindIII digested gDNA was probed with the above-mentioned flanking region fragments. The GFP fluorescence signal from each transplastomic strain was confirmed under UV light. The photo was taken 2 months after germination. The bar represents 0.5 cm. Western blot analysis for concentration determination using GFP standard protein. Lyophilized (10 mg) and fresh leaf material (100 mg) was extracted with 300 μL extraction buffer. 1X represents 1 μL of homogenate resuspended in extraction buffer at a ratio of 100 mg to 300 μL. Amount of GFP fusion protein in fresh (F) and lyophilized (L) leaves. Data are the mean ± SD of 3 independent experiments. 2A-2B. Efficiency of oral delivery and biodistribution of GFP fused with different tags. GFP levels in serum (FIG. 2A) and tissue (FIG. 2B) in mice (N = 6 / group) fed with leaf material expressing CTB-GFP, PTD-GFP and DCpep-GFP. Adult mice were orally administered for 3 consecutive days with leaf material from genetically modified tobacco plants in an amount adjusted to the GFP expression level. The control group (N = 6) remained unsold. Blood samples were taken 2 hours and 5 hours after the last gavage, mice were sacrificed, and tissue samples were collected for protein isolation. GFP concentrations in serum and tissues were measured by ELISA. Data are shown as mean ± SEM. Statistical significance was determined by paired student t-test, and p values less than 0.05 were considered significant. * P <0.05, ** P <0.01, *** P <0.05, or P <0.01 (CTD, PTD and DCpep are untreated as controls) 3A-3L. Visualization of GFP in mouse ileum and liver cells after oral delivery of plant cells. (Left panel) GFP delivery to the small intestine. Cross sections stained with anti-GFP (green signal; Alexa Fluor 488), UEA-1 (among other cells, stain M cells, red signal, rhodamine), and DAPI (nuclear stain, blue) are shown. (FIGS. 3A-3C). PTD-GFP delivery. (FIG. 3B) No primary antibody (NC: negative control). (FIGS. 3D-3E) CTB-GFP delivery. (FIG. 3F) DCpep delivery. Original magnification: 200 × (insertion in FIGS. 3A, B, DF, C) or 40 × (FIG. 3C). (Right panel) GFP staining shown in frozen section of liver, same exposure time as imaging. Primary antibody: 1: 1000 rabbit anti-GFP antibody and secondary antibody: Alexa Fluor 488 donkey anti-rabbit IgG were used for GFP staining. (G, I and K) Liver sections of mice fed non-transformed, lyophilized plant cells. (FIGS. 3H, J and L) Liver sections from mice fed with lyophilized plant cells expressing DCPep-GFP (FIG. 3H), PTD-GFP (FIG. 3J) and CTB-GFP (FIG. 3L). GFP signal. Original magnification: 100 times. 4A-4F. Properties of purified GFP fusion protein. (FIG. 4A) Quantification of purified GFP fusion protein and coomassie staining and fluorescence image. A densitometric assay with Western blot images was performed using known amounts of GFP standard protein to quantify the purified tag-fused GFP protein. The purified protein was run on SDS-PAGE and immunoprobed with anti-GFP antibody. The load was as shown. Purity was calculated as a percentage of the amount detected in the immunoblot assay relative to the total loading. (FIG. 4B) Coomassie staining of purified GFP-tagged protein. M: protein molecular weight marker; lane 1: PTD-GFP (10 μL, 2.37 μg): lane 2: Dcpep-GFP (40 μL, 3.12 μg): lane 3: CTB-GFP (10 μL, 32.8 μg): and Lane 4: GFP (400 ng). (FIG. 4C) Non-denaturing SDS-PAGE of purified GFP fusion protein to measure GFP fluorescence. Lane 1 (PTD-GFP 10 μl, 9.17 μg TSP load), Lane 2 (DCpep-GFP 15 μl, 4.6 μg TSP load) and Lane 3 (CTB-GFP 20 μl, 33 μg TSP load). (FIGS. 4D and 4E) Purified GFP-tagged proteins were examined for their binding affinity to the GM1 receptor. Anti-CTB (Figure 4D) and anti-GFP (Figure 4E) antibodies were used to detect the interaction between GM1 and the GFP fusion protein. The amount of protein used in the assay is as follows. CTB: 10 pg; CTB-GFP: 1.25 ng; PTD-GFP: 10 ng; DCpep-GFP: 10 ng; GFP: 10 ng: and UT, untransformed wild type total protein: 100 ng. (FIG. 4F) Undenatured Tris-Tricine PAGE of purified CTB-GFP to determine pentamer structure. The purified pentameric structure of CTB-GFP was immunoprobed using an anti-CTB antibody (1 / 10,000). The amount of CTB-GFP loaded is shown in the figure. 5A to 5B. Uptake of GFP fused with different tags by human immune and non-immune cells. (FIG. 5A) Transfer of purified GFP fusion protein in human cell lines. Cultured human dendritic cells (DCs), B cells, T cells and mast cells 2 × 10 ⁴ cells were incubated with the purified GFP fusion. CTB-GFP (8.8 μg / 100 μl PBS), PTD-GFP (13 μg / 100 μl PBS), DCpep-GFP (1.3 μg / 100 μl PBS) and commercially available standard GFP (2.0 μg / 100 μl PBS), respectively. Incubated for 1 hour at 37 ° C. After PBS washing, B, T and mast cell pellets were stained with DAPI diluted 1: 3000 and fixed with 2% paraformaldehyde. The cells were then sealed on slides and examined by confocal microscopy. Viable DCs were stained with Hoechst diluted 1: 3000 and detected directly under a confocal microscope. For 293T, pancreatic cells (PANC-1 and HPDE) and macrophage cells, 8-well chamber slides were used for cell culture overnight at 37 ° C. Purified CTB-GFP (8.8 μg / 100 μl PBS), PTD-GFP (13 μg / 100 μl PBS), DCpep-GFP (1.3 μg / 100 μl PBS) and commercial standard GFP (2.0 μg / 100 μl PBS), respectively. Incubated for 1 hour at 37 ° C., washed with PBS, and stained nuclei with 1: 3000 DAPI. (FIG. 5B) Nuclear localization of PTD-GFP in human pancreatic duct epithelial cells (HPDE). Green fluorescence indicates GFP expression. Blue fluorescence indicates nuclear labeling with DAPI. The image was observed at a magnification of 100 times. The scale bar represents 10 μm. All imaging studies were analyzed in triplicate. 6A and 6B. Incorporation of GFP fused with different tags by different human somatic and stem cell types. (FIG. 6A) Human pancreatic cells (PANC-1 and HPDE), human periodontal ligament stem cells (HPDLS), maxilla mesenchymal stem cells (MMS), human head and neck flats Epithelial cells (human head and neck squamousomas cells) (SCC), retinal pigment epithelial cells (RPE), gingival-derived mesenchymal stromal cells (gingivemcell cells) , Adult gingival keratinocytes (adult gingival keratinocytes) (AG K) and purified fusion of CTB-GFP, PTD-GFP, DCpep-GFP and GFP-Proteglin 1 (GFP-PG1) and GFP-Retrocyclin 101 (GFP-RC101) in osteoblasts (OBC) In vitro assessment of protein transformation using confocal microscopy. The human cell lines PANC-1, HPDE, HPDLS, MMS, SCC, RPE, GMSC, AGK and OBC 2 × 10 ⁴ cells were cultured overnight at 37 ° C. in 8-well chamber slides (Nunc). Subsequently, it was incubated with the purified GFP fusion protein. : CTB-GFP (8.8 μg), PTD-GFP (13 μg), DCpep-GFP (1.3 μg), GFP-PG1 (1.2 μg), GFP-RC101 (17.3 μg), respectively, 100 μl of FBS Were supplemented with 1% PBS and incubated at 37 ° C. for 1 hour. After washing the wells 3 times with PBS, the cells were stained with 1: 3000 DAPI for 10 minutes at room temperature and then fixed with 2% paraformaldehyde for 10 minutes at room temperature. For negative control wells, cells were incubated with commercial GFP (2 μg) in PBS containing 1% FBS or untreated. After 1 hour of incubation, the cells were washed 3 times with PBS. All fixed cells were imaged using a confocal microscope. Green fluorescence indicates GFP expression. Blue fluorescence indicates nuclear labeling with DAPI. The image was observed with a 100 × objective lens. The scale bar represents 10 μm. All imaging studies were analyzed in triplicate. (FIG. 6B) Human pancreatic cells (PANC-1 and HPDE), human periodontal ligament stem cells (HPDLS), epithelial-mesenchymal stem cells (MMS), human head and neck squamous cells (SCC), CTB-GFP, PTD-GFP, purified fusion proteins in retinal pigment epithelial cells (RPE), gingival-derived mesenchymal stromal cells (GMSC), adult gingival keratinocytes (AGK), and osteoblasts (OBC) Evaluation of in vitro transformation of DCpep-GFP, PG1-GFP and antimicrobial peptides (PG1-GFP and RC101-GFP) using a confocal microscope.

Detailed Description of the Invention Targeted oral delivery of GFP fused to GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated.
The presence of GFP ⁺ intact plant cells between the ileal villi confirms their protection in the digestive system from acid / enzymes.
Efficient delivery of GFP to intestinal epithelial cells by PTD or CTB, and efficient delivery of GFP to M cells by all these fusion tags confirms GFP uptake in the small intestine.
The PTD fusion delivered GFP more efficiently to most tissues or organs than the other two tags.
GFP was efficiently delivered to the liver by all fusion tags, possibly via the gut-liver axis.
In a confocal imaging study of human cell lines using purified, GFP fused with different tags, the GFP signal of DCpep-GFP was detected only in dendritic cells.
PTD-GFP was detected only in kidney cells or pancreatic cells, but not in immunoregulatory cells (macrophages, dendritic cells, T, B, or mast cells).
In contrast, CTB-GFP was detected in all cell types tested, confirming the ubiquitous presence of the GM1 receptor.

  Such low-cost oral delivery of protein drugs to serum, immune system or non-immune cells dramatically reduces their cost by eliminating very expensive fermentation, protein purification, refrigeration / transport And should improve patient compliance.

Definition:
As used herein, the term “administering” or “administration” of a drug, medicament or peptide to a subject introduces a compound that performs its intended function into the subject. Or any route of delivery. Administration or administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal or topical. it can. Administration or administration includes self-administration and administration by others.

As used herein, the terms “disease”, “disorder” or “complication” refer to any deviation from a normal state in a subject.
As used herein, the terms “effective amount”, “effective amount” and the like refer to a single dosage and time required to achieve a desired result. Means an effective amount of

  As used herein, the terms “inhibit” or “treat” are exposed to or susceptible to a disease state, but have not yet experienced symptoms of the disease state, or In subjects not shown, does not exacerbate or develop clinical symptoms of the disease state, eg, inhibit the onset of the disease.

  As used herein, the term “expression” in the context of a gene or polynucleotide includes transcription of the gene or polynucleotide into RNA. The term can also include, but is not necessarily, the subsequent translation of RNA into polypeptide chains and their incorporation into proteins.

  A “transformed plant” refers to a plant whose genome has been altered by the introduction of at least one heterologous nucleic acid molecule.

As used herein, “nucleic acid” or “nucleic acid molecule” refers to either a single-stranded or double-stranded DNA or RNA molecule and, if single-stranded, linear or circular Refers to any complementary sequence molecule.
In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule can be described herein according to the usual convention of providing a sequence in the 5 'to 3' direction.
With respect to the nucleic acids of the invention, the term “isolated nucleic acid” may be used.
When applied to DNA, the term refers to a DNA molecule that is separated from sequences that are immediately contiguous in the naturally occurring genome of the organism from which it is derived. For example, an “isolated nucleic acid” can include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

  When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that is well separated from other nucleic acids that will be involved in its natural state (ie, in a cell or tissue).

  An “isolated nucleic acid” (either DNA or RNA) further represents a molecule that is produced directly by biological or synthetic means and separated from other components present during its production. Also good.

  As described in the University of Wisconsin GCG software program, the terms “percent similarity”, “percent identity” and “percent homology” are used when referring to a particular sequence.

  The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of a given material (eg, nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% and most preferably 90-95% by weight of a given compound. Purity is measured by methods appropriate for a given compound (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

  A “replicon” is any genetic element that is capable of replicating substantially under its own control, such as a plasmid, cosmid, bacmid, phage or virus. The replicon may be either RNA or DNA and may be single stranded or double stranded.

  A “vector” is any vehicle to which another gene sequence or element (either DNA or RNA) is added to cause replication of the added sequence or element.

  “Expression operon” refers to a nucleic acid segment that can have transcriptional and translational control sequences, such as a promoter, enhancer, translation initiation signal (eg, ATG or AUG codon), polyadenylation signal, terminator, etc., and a host Facilitates expression of a polypeptide coding sequence in a cell or organism.

As used herein, the term “oligonucleotide” refers to the sequences, primers and probes of the present invention, and a nucleic acid molecule comprising two or more ribonucleotides or deoxyribonucleotides, preferably three or more nucleic acid molecules. Is defined as
The exact size of the oligonucleotide will depend on various factors, as well as the particular application and use of the oligonucleotide.

The term “specifically hybridizes” allows such hybridization under certain conditions commonly used in the art (sometimes referred to as “substantially complementary”). Refers to the association between two single stranded nucleic acid molecules of sufficient complementary sequence.
In particular, the term includes hybridization of oligonucleotides having substantially complementary sequences contained within single-stranded DNA or RNA molecules of the invention, and single-stranded nucleic acid and oligonucleotides of non-complementary sequences. Refers to substantial elimination of hybridization.

  The term “promoter region” refers to the 5 ′ regulatory region of a gene (eg, 5′UTR sequence (eg, psbA sequence, promoter (eg, universal Prnn promoter or psbA promoter endogenous to the plant to be transformed) and desired Enhancer element).

  As used herein, the term “reporter”, “reporter system”, “reporter gene” or “reporter gene product” refers to a reporter signal that is readily measurable when a nucleic acid is expressed (eg, An operational genetic system that includes a gene encoding a product that produces a biological assay, immunoassay, radioimmunoassay, or colorimetric calorimetric, fluorescent fluorgenic, chemiluminescent chemiluminescent or other method Means.

Nucleic acids can be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and act on regulatory elements necessary for expression of the reporter gene product Connected as possible.
The required regulatory elements vary depending on the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but include elements such as promoters, enhancers, translation control sequences, poly A addition signals, transcription termination signals, etc. It is not limited to.

  The terms “transform”, “transfect”, “transduce” refer to any method or means by which a nucleic acid is introduced into a cell or host organism, and Can be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG fusion, and the like.

  The term “selectable marker gene” confers a selectable phenotype when expressed, such as antibiotic resistance, on transformed cells or plants. Refers to a gene.

The term “operably linked” means that the regulatory sequences necessary for the expression of the coding sequence are placed in the DNA molecule at an appropriate position with respect to the coding sequence to effect the expression of the coding sequence. means.
This same definition may apply to the placement of transcription units and other transcription control elements (eg, enhancers) in an expression vector.

  The term “DNA construct” refers to a gene sequence that is used to transform a plant and produce progeny of the transformed plant. These constructs can be administered to plants with viral or plasmid vectors. However, most preferred for use in the present invention are plastid transformation vectors.

  Other delivery methods such as Agrobacterium T-DNA mediated transformation and transformation using biolistic processes are also considered to be within the scope of the present invention.

  Transformed DNA is described in "Current Protocols in Molecular Biology", Frederick M. et al. Adjusted according to standard protocols as described in Ausubel et al., John Wiley & Sons, 1995.

  The phrase “double-stranded RNA-mediated gene silencing” thereby causes the target gene expression to form a double-stranded RNA structure with the target gene encoding mRNA that is subsequently degraded. It refers to a process that is suppressed in plant cells through the introduction of a nucleic acid construct encoding the molecule.

  When referring to a particular nucleotide or amino acid, the phrase “consisting essentially of” means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, this phrase includes the sequence itself and molecular modifications that do not affect the basic and novel properties of the sequence.

The terms “tag”, “tag sequence” or “protein tag” provide additional utility or confer useful properties when added to another sequence. Refers to a chemical moiety of either an oligonucleotide, or more preferably a peptide or other chemical, such that it specifically targets the protein of interest to the desired cell type. Such tags can also be useful for isolating and purifying fusion proteins containing them.
A protein tag, as described herein or as commonly used in the art, can be added to either the amino terminus or the carboxy terminus of the protein of interest.

  “Immune response” means any reaction produced by an antigen, such as a viral or plant antigen, in a host with a functioning immune system. The immune response may be humoral in nature (ie involving the production of immunoglobulins or antibodies) or cellular in nature (various types of B and T lymphocytes, dendritic cells, Macrophages, antigen-presenting cells, etc.), or both. The immune response can also include the production or refinement of various effector molecules such as cytokines, lymphokines. The immune response can be measured both in vitro and in various cellular or animal systems.

  An “antibody” or “antibody molecule” is any immunoglobulin (including antibodies and fragments thereof) that binds a specific antigen. Some of the drugs listed in Table 4 are antibodies. The term includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and bispecific antibodies. As used herein, an antibody or antibody molecule is an intact immunoglobulin molecule and those known in the art such as Fab, Fab ′, F (ab ′) 2 and F (v). By both immunologically active portions of immunoglobulin molecules such as some are meant.

As used herein, a fusion peptide increases the ability of a protein to enter a cell by fusing with the cell membrane without the need for specific receptors. Other fusion proteins can target cellular receptors. Certain fusion proteins specifically target immune cells. Others, like DC peptides, target dendritic cells.
Some fusion proteins are fairly non-specific and can be used to deliver therapeutic proteins to various cell types of interest.

  Plant remnants include one or more molecules (including but not limited to proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, etc.) derived from the plant in which the protein of interest is expressed. obtain. Thus, a composition associated with whole plant material (eg, all or part of a plant leaf, stem, fruit, etc.) or crude plant extract will result in a high concentration of plant residue, and one or more detectable plants. It will certainly include a composition containing the purified protein of interest with a residue. In certain embodiments, the plant residue is rubisco.

  In another embodiment, the invention treats a disease through administration of a therapeutic fusion protein produced in a plant chloroplast comprising a tag that delivers the therapeutic fusion protein to a target cell or tissue of interest. Or relates to an administrable composition for prevention. The composition comprises a therapeutically effective amount of the fusion protein expressed by the plant and plant residue.

Methods, vectors and compositions for transforming plants and plant cells are taught, for example, in WO 01/72959, WO 03/057834; and WO 04/005467. ing. WO 01/64023 discusses the use of marker-free gene constructs.
Proteins expressed according to particular embodiments taught herein can be used in vivo by administering to a subject, human or animal in a variety of ways. The pharmaceutical composition can be administered orally or parenterally, ie subcutaneously, intramuscularly or intravenously, although oral administration is preferred.
A list of therapeutic proteins of interest is provided in Example II.

Oral compositions produced according to embodiments of the present invention can be administered by consumption of manufactured foods using transformed plants that produce plastid-derived therapeutic fusion proteins. The edible part of the plant or a part thereof is used as a food ingredient.
The therapeutic composition can be formulated in a classic manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the composition can be administered in the form of tablets, capsules, granules, powders, etc. with at least one vehicle such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. The preparation may be emulsified.
The active immunogenic or therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents or adjuvants.
In a preferred embodiment, the edible plant, juice, grain, leaf, tuber, stem, seed, root or other plant part of the pharmaceutical transformed plant is ingested by a human or animal and is therefore therapeutic or immune to the disease. Provides a very inexpensive means. In certain embodiments, plant material comprising chloroplasts capable of expressing a therapeutic fusion protein (eg, lettuce, tomato, carrot, low nicotine tobacco material, etc.) is homogenized and encapsulated. In one particular embodiment, an extract of lettuce material is encapsulated. In another embodiment, the lettuce material is powdered prior to encapsulation. In another embodiment, for convenience of administration, the composition may be provided in the juice of a transformed plant.
For this purpose, the plants to be transformed are preferably selected from edible plants consisting in particular of tomatoes, carrots and apples, which are usually consumed in the form of juices.

According to another embodiment, the present invention relates to a transformed chloroplast genome transformed with a vector comprising a heterologous gene expressing a therapeutic fusion protein or peptide as disclosed herein.
References to protein sequences herein are at least 12, 15, 25, 50, 75 selected from known full-length amino acid sequences, as well as such amino acid sequences or biologically active variants thereof. , 100, 125, 150, 175, 200, 225, 250 or 265 consecutive amino acids. Typically, a polypeptide sequence relates to a known human version of that sequence.

Biologically active variants are characterized by upregulation of immunosuppressive cytokines (such as IL10 and IL4) and serum antibodies (such as IgG1) in the case of oral tolerance. Refers to a variant that activates T cells and / or induces a Th2 cell response, or a variant that maintains the natural function of the protein if one desires the natural function of the protein.
Preferably, naturally or non-naturally occurring polypeptide variants are at least about 55,60,65 or 70, preferably about 75,80,85,90, relative to the full length amino acid sequence or fragment thereof. 96, 96 or 98% have the same amino acid sequence.
The percent identity between the putative polypeptide variant and the full length amino acid sequence is determined using the Blast2 alignment program (Blosum62, Expect 10, standard gene code).
Changes in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one-to-one amino acid substitutions. If substituted amino acids have similar structural and / or chemical properties, they are naturally conservative. Examples of conservative substitutions are substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, or substitution of threonine with serine.
An amino acid insertion or deletion is a change in or within an amino acid sequence.
They are typically in the range of about 1-5 amino acids.
Guidance for determining which amino acid residues can be substituted, inserted or deleted without losing the biological or immunological activity of the polypeptide is well known in the art, such as DNASTAR software. Can be found using a computer program.

Whether an amino acid change results in a biologically active therapeutic fusion polypeptide is readily determined, for example, by assaying for natural activity, as described in the specific examples below. Can do.
Reference herein to a gene sequence means a single-stranded or double-stranded nucleic acid sequence and includes the coding sequence of the polypeptide of interest or the complement of the coding sequence.

A degenerate nucleic acid sequence encoding a homologous nucleotide sequence that is at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to a polypeptide as well as cDNA is It can be used according to the teachings of polynucleotides in the specification.
The percent sequence identity between the sequences of two polynucleotides was determined using an affine gap search (gap open penalty of -12) and gap extension penalty of -2 (gap extension penalty of -2.). It is determined using a computer program such as ALIGN which employs the FASTA algorithm.
Complementary DNA (cDNA) molecules, species homologues, and variants of nucleic acid sequences that encode biologically active polypeptides are also useful polynucleotides.
Variants and homologs of the above nucleic acid sequences are also useful nucleic acid sequences.
Typically, homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides with known polynucleotides under stringent conditions, as is known in the art.
For example, use the following cleaning conditions:
2 × SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, twice each at room temperature for 30 minutes each;
Next, 2 × SSC, 0.1% SDS, once at 50 ° C. for 30 minutes;
Then 2XSSC, twice at room temperature for 10 minutes each
At most, homologous sequences containing about 25-30% base pair mismatch can be identified.

More preferably, the homologous nucleic acid strand comprises 15-25% base pair mismatch, even more preferably 5-15% base pair mismatch.
Polynucleotide species homologs referred to herein can also be identified by making appropriate probes or primers and screening cDNA expression libraries.
It is well known that the Tm of double-stranded DNA decreases by 1 to 1.5 ° C. every time the homology decreases by 1% (Bonner et al., J. Mol. Biol. 81, 123 (1973)). . Polynucleotides of interest, or their complements subject to stringent hybridization and / or wash conditions, are also useful polynucleotides. For example, in Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition, 1989, pages 9.50 to 9.51, stringent wash conditions are well known and understood in the art, And disclosed,

Typically, for stringent hybridization conditions, a combination of temperature and salt concentration should be selected that is about 12-20 ° C. lower than the calculated T _m of the hybrid under study. The T _m of the hybrid between the polynucleotide of interest or its complement and a polynucleotide sequence that is at least about 50, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence is, for example, Using the Bolton and McCarthy equations, Proc. Natl. Acad. Sci. USA 48, 1390 (1962), can be calculated:
Tm = 81.5 ° C.−16.6 (log 10 [Na +]) + 0.41 (% G + C) −0.63 (% formamide) −600 / l),
Where l = length of the hybrid in base pairs.
Stringent wash conditions include, for example, 4 × SSC at 65 ° C., or 50% formamide at 42 ° C., 4 × SSC, or 0.5 × SSC, 0.1% SDS at 65 ° C.
Highly stringent wash conditions include, for example, 0.2 × SSC at 65 ° C.
In order to facilitate the practice of the present invention, the following materials and methods are provided.

  Generation of transplastomic lines expressing different tagged GFP fusion proteins

Transplastic plants that express CTB-GFP and PTD-GFP were generated as described in previous studies [6, 34]. DC-specific peptides are described in Ph. D. Identified from screening. A 12-mer phage display library [26] is conjugated to the C-terminus of GFP and cloned into a chloroplast transformation vector, and trans expressing DCpep-GFP. A plastomic line was created and a homoplasmic line (homoplasmic lines, identical internal structural strain) was confirmed using the Southern blot assay described previously [35].
The expression of GFP-tagged protein was confirmed by visualizing green fluorescence from the leaves of each construct under UV irradiation.

Lyophilized Harvested mature leaves were stored at −80 ° C. and lyophilized using a lyophilizer (Genesis 35XL, VirTis SP Scientific), thereby freezing and crushed, small pieces of leaves Was sublimated under vacuum conditions (400 mTorr) for 3 days at −40 ° C. to 25 ° C. under conditions where the chamber temperature was gradually increased.
The lyophilized leaves were then ground in a coffee grinder (Hamilton Beach) three times (10 seconds each) at maximum speed.
The powdered plant cells were stored at room temperature in an airtight and moisture-free state using silica gel.

Quantification of GFP fusion protein and GM1 binding assay Densitometry (densitometry concentration measurement) assay and GM1 ELISA assay for quantification of GFP fusion protein are GFP standard protein (Vector laboratories MB-0752-100) and mouse monoclonal anti-GFP. Performed according to previous method [14] except using antibody (EMD MILLIPORE MAB3580). To identify the pentameric structure of CTB-GFP, a non-denaturing tris-tricine gel was performed according to previous studies [36].

Purification of tag-fused GFP protein Protein purification of GFP fusion peptides, PTD-GFP and DCpep-GFP was performed by the organic extraction / FPLC-based method described in [37] above.
Approximately 200 mg of lyophilized plant cells was added to 10 ml of plant extraction buffer (100 mM NaCl, 10 mM EDTA, 200 mM Tris-Cl pH 8.0, 0.2% Triton X, 400 mM sucrose (Sucrose), 2% v / v PMSF, (1 tablet of protease inhibitor contained in a total volume of 10 ml) was homogenized. After sonication, the homogenate was spun down and the supernatant was collected.
The supernatant was transferred to a 50 ml falcon tube and subjected to organic extraction as previously done [37].
The plant extract was treated with saturated ammonium sulfate so that the final concentration in the extract was 70%. Then a quarter of the total extract of 100% ethanol was added, mixed vigorously for 2 minutes, and then spun down.
The resulting organic phase (upper phase) was collected in a new 50 ml falcon tube. To the remaining aqueous phase, 1/16 of 100% ethanol was added and shaken vigorously for 2 minutes and then spun down again.
The organic phases from both spins are pooled together and 5M NaCl is added 1/3 of the total volume and n-butanol 1/4 of the resulting volume and shaken vigorously for 2 minutes. Rotation settled.
The organic extract layer (lower phase) obtained at the bottom of the tube was collected and then desalted by passing through a 7 KDa MWCO desalting column (Thermo scientific zeba spin column 89893). The organic extract was loaded onto a desalting column and spun down according to the manufacturer's instructions.
The desalted organic extract (approximately 5 ml volume) was then loaded onto an FPLC column (LKB-FPLC purification system, Pharmacia; 48 mL column volume). During the purification process, the samples were washed with 3.5 column volumes of buffer A (set to 10 mM Tris HCl, 10 mM EDTA and 291 gm ammonium sulfate set, pH 7.8) saturated with 20% ammonium sulfate.
The column was then stepped with Buffer B (set to 10 mM Tris HCl, 10 mM EDTA sulfate, pH 7.8) to elute the GFP fusion.
Protein was detected by measuring absorbance at 280 nm, corresponding to a single peak plotted on the recorder. The fraction corresponding to the peak was collected in a single tube with a total volume of 9 ml.
The purified fraction was then dialyzed 3 times with 2 L of 0.01 × PBS and then lyophilized (Labconco lyophilizer). The lyophilized purified GFP fusion was then quantified by Western blot / densitometry.
For purification of CTB-GFP, lyophilized leaf material (400 mg) contains 20 ml extraction buffer (50 mM Na-P, pH 7.8; 300 mM NaCl; 0.1% Tween-20; 1 tb EDTA) Resuspended in a protease inhibitor cocktail).
The resuspension was sonicated and then centrifuged at 10,000 rpm, 4 ° C. for 10 minutes. The supernatant was mixed with 1 ml of His60 Ni resin (Clonetech, 635657) and purified according to the manufacturer's instructions.

Purity measurement and Coomassie staining
A Bradford assay was used to quantify the total protein purified from each GFP-tagged fraction and then a densitometric assay was performed to determine the amount of GFP fusion protein in the fraction as described in the quantification section. Quantified.
The purity was then assessed by calculating the percentage of the amount of GFP fusion protein relative to the total amount of protein obtained from the Bradford assay.
Non-denaturing SDS-PAGE was also performed to examine the fluorescence of the GFP fusion protein by passing through a 10% SDS gel under non-denaturing conditions.

Assessment of GFP expression The presence of GFP in serum and tissues was quantified by our in-house GFP ELISA. As in our previous report [6, 16], blood and tissue samples were taken and serum stored at −80 ° C. 2 and 5 hours after the last oral gavage.
Tissues were homogenized in RIPA buffer and supernatants were collected for GFP ELISA assays. Our in-house ELISA protocol was established and the standards were calibrated based on the GFP ELISA kit (AKR121; Cell Biolab).
Briefly, 96-well Maxisorp plates (Nunc) were coated overnight at 4 ° C. with goat polyclonal GFP antibody (2.5 mg / ml, Rockland) in coating buffer (pH 9.6). Were blocked with PBS containing 3% BSA for 2 hours at 37 ° C.
Serial 2-fold dilutions of serum in PBS containing 1% BSA were added in duplicate and incubated overnight at 4 ° C.
Plates were then incubated overnight at 4 ° C. with biotin-fused rabbit polyclonal anti-GFP 1: 5000 (Rockland), followed by addition of 1: 2000 diluted streptavidin peroxidases (Rockland). .
After further incubation at 37 ° C. for 1 hour, the plate was washed and the substrate solution was added and incubated at room temperature for 10 minutes.
The reaction was stopped by adding 100 μl of 2N sulfuric acid per well, and the absorbance was measured at 450 nm using an ELISA reader. Results are shown as mean ± SEM.

Mice and Oral Delivery Experiments Eight week old female C57BL / 6 mice (Jackson Laboratories, Bar Harbor, MI) were randomly divided into 4 groups (6 mice per group) and lyophilized bioencapsulated. CTB-GFP, PTD-GFP and DCpep-GFP plant cells were orally administered 20 mg / mouse / day for 3 consecutive days. All lyophilized material was suspended in 200 μl PBS. On day 3, blood samples were taken 2 hours and 5 hours after the last oral gavage.
At 5 hours, all mice were sacrificed and organs (liver, kidney, lung, brain, tibialis muscle (TA)) were harvested and stored at −80 ° C.
A control group (n = 6) received untransformed lyophilized plant cells. Mice were housed at the University of Florida and University of Pennsylvania animal facilities under controlled humidity and temperature conditions and experiments were performed according to animal experimentation and use committee guidelines.

Immunofluorescent staining (Immunofluorescent staining)
As described previously [8, 16], C57BL / 6 mice were orally fed GFP-expressing plant cells twice at 2-hour intervals. Two hours after the last feeding, the mice were sacrificed and the liver and intestine were removed. The intestine was cut longitudinally and washed with PBS, then rolled up and fixed with 4% paraformaldehyde overnight at 4 ° C. Liver tissue was similarly fixed.
Subsequently, the fixed tissues were further incubated at 30C in 30% sucrose in PBS and embedded in OCT. Serial sections were cut to a thickness of 10 μm.
For analysis of GFP expression, sections were permeabilized with 0.1% Triton X-100 and blocked with 5% donkey serum in PBS for 30 minutes, followed by 1: 1000 (ab290 , Abcam) with a rabbit anti-GFP antibody at 4 ° C. overnight. The sections were then incubated with Alexa Fluor 488 donkey anti-rabbit IgG (Jackson ImmunoResearch) for 30 minutes or with rhodamine-labeled Ulex europaeus agglutinin (UEA-1; Vector Labs; 10 μg / mL) for 10 minutes, Washed and mounted with or without DAPI (4,6 diamidino-2-phenylindole). Images were captured using a Nikon Eclipse 80i fluorescent microscope and a Retiga 2000R digital camera (QImaging) and analyzed with Nikon Elements software.

Uptake of purified tag-fused GFP protein by human cell lines To determine uptake of three tags, CTB, PTD and DC peptide in immunoregulatory cells, mature dendritic cells, human T cells (Jurkat cells), human B cells (BCBL1), macrophage cells (mφ), mast cells and non-immunoregulatory cells, human kidney cells (293T), human pancreatic epithelial-like cancer cells (PANC-1) and human pancreatic duct epithelial cells (HPDE) and The purified tag fusion protein was used for in vitro transformation.
Cells (2 × 10 ⁴ ) were combined with purified CTB (8.8 μg), PTD (13.5 μg) and DCpep (1.3 μg) fusion proteins, respectively, in 1% PBS supplemented with 100 μl FBS. And incubated at 37 ° C. for 1 hour.
After washing with PBS, the cell pellet was stained with DAPI diluted 1: 3000 and fixed with 2% paraformaldehyde for 10 minutes at room temperature. Cells were then sealed on a glass slide with cytosol and examined by confocal microscopy.
For live image studies, after incubation with purified GFP fusion protein, dendritic cells were mounted on glass-bottomed microwell dishes (MatTek) and observed under a confocal microscope followed by nuclear staining with DAPI did.
For 293T, PANC-1 and macrophage cells, cells were cultured overnight at 37 ° C. in 8-well chamber slides (Nunc), followed by purified CTB-GFP, PTD-GFP and DCpep-GFP (above The same) at 37 ° C. for 1 hour.
After washing the wells with PBS, cells were stained with 1: 3000 DAPI and fixed with 2% paraformaldehyde for 10 minutes at room temperature.
In the negative control group, cells were incubated with commercial GFP (2 μg) or untreated in PBS containing 1% FBS.
After 1 hour of incubation, the cells were washed once with PBS. Viable, non-fixed cells were imaged using a confocal microscope.

To determine the uptake efficiency of purified GFP fusion protein in different human cell lines, the number of cells showing GFP signal was counted and expressed as a percentage of the total number of cells observed.
For each cell line in three independent samples, a total of 15-20 images were recorded under a confocal microscope at 100x magnification.

  The following examples are provided to illustrate specific embodiments of the invention. These do not limit the invention in any way.

Example I
Incorporation of GFP fused with different tags by human cells In this study, we investigated the oral administration of plant cells expressing GFP fused with different tags in chloroplasts, and investigated cell targeting and its biodistribution. evaluate.
Delivery of PTD, DCpep and CTB fusions across the intestinal epithelium, utilizing different routes, results in systemic delivery, biodistribution, and most importantly, uptake of different patterns by non-immune or immunoregulatory cells. Hesitate.
These peptides can be used to deliver therapeutic proteins to serum, immunoregulatory cells or specific tissues.

Three separate transmucosal carriers were tested.
The interaction between CTB pentamers and GM1 receptors is well studied, as shown in FIG. 1A. The pentasaccharide structure of the GM1 receptor interacts with the amino acids of CTB via hydrogen bonding [38]. In contrast, the structural mechanisms shown for PTD (FIG. 1A) or DCpep (FIG. 1A) have not been fully studied.
The secondary structure of cell-penetrating peptides (CPP) has been studied by circular dichroism (CD). Such peptides interact with negatively charged phospholipid vesicles that result in induction of secondary structure. In such studies, small unilamellar vesicles (SUV) are used. However, it is difficult to define the exact role of any secondary structure of the CPP with respect to the translocation process. CPP has been shown to change structure from an α helix to a beta sheet, depending on experimental conditions, even in a simple model system. They are therefore described as “chameleons” that change their structure and adapt rapidly to the membrane environment (39).
Therefore, in our study, secondary structure was not studied, but I-TASSER (interactive threading assembly refinement program) was used to predict the computational 3-D structure [40].

In FIG. 1A, a representative model is selected based on the calculation of parameters such as confidence score (C-score), high resolution model with root mean square deviation (RMSD) value, and template modeling score (TM-score). It was.
For example, if the TM-score is greater than 0.5, which evaluates the topological similarity between the initial I-TASSER model and the corresponding structure of the protein databank (PDB) library, the predicted topology is correct [ 40]. From the prediction model, the PTD TM-score was 0.60 ± 0.14 and the DCpep TM-score was 0.58 ± 0.14. In the predicted structure, PTD has a helical structure, while DCpep shows a loop structure. As expected, there is no similarity between the two peptides.
All three tags were fused to green fluorescent protein (smGFP) to evaluate its efficiency and specificity.
CTB (MIKLKFGVFFTVLLSASAHGTPQNITDLCAEYHNTQIHTNLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLEAKVEKLCVWNNKTPHA
Sixteen amino acids (RHIKIWFQNRRMKWKK; SEQ ID NO: 3) derived from pancreas and duodenal homeobox factor-1 (PDX-1) [41] were fused with GFP at the N-terminus and in this study PTD-GFP Call it.
Additional localization signals are required for nuclear targeting.
The six amino acids of 16aa-PTD (RH, RR, and KK) are important for nuclear localization of PDX-1 (42).
A human dendritic cell specific peptide ligand (FYPSYHSTPQRP (SEQ ID NO: 4)), identified from screening of a 12-mer phage display library [26], is fused to the C-terminus of GFP.
Both PTD and DC-peptide were engineered to have no furin cleavage site to study invasion and tissue distribution.
As described in the Materials and Methods section, all these three fusion constructs were cloned into a chloroplast transformation vector (pLD) that was used to transform chloroplasts.

To produce plants that express GFP fusion proteins, tobacco chloroplasts were transformed using a biolistic particle delivery system.
As seen in FIG. 1B, each tag-fused GFP is stabilized by the same regulatory sequences—the 5 ′ UTR controlled by the psbA promoter and light, and the transcribed mRNA is stabilized by the 3 ′ psbA UTR. Driven. The psbA gene is the most highly expressed chloroplast gene and, therefore, psbA regulatory sequences have been used for transgene expression in our laboratory [7,35].
To facilitate integration into the chloroplast genome of the expression cassette, the two flanking sequences of the isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase (trnA) genes are identical to the native chloroplast genome sequence. Adjacent to an expression cassette.
Emerging shoes from selection medium for specific integration of the transgene cassette in the spacer region of trnI and trnA, and then containing flanking sequences of trnI and trnA Southern blot analysis using labeled probes examined the transformation of all chloroplast genomes in each plant cell (the absence of untransformed wild-type chloroplast genomes). (FIG. 1C).
As seen in FIG. 1C, HindIII-digested gDNA from three lines of each GFP plant was 7.06 for CTB-GFP, PTD-GFP, and DCpep-GFP, respectively, when hybridized with the probe. , 6.79 and 6.78 kbp, the absence of large transformed DNA fragments and the untransformed smaller fragment (4.37 kbp).
Therefore, stable integration of three different GFP expression cassettes and homoplasmy of the chloroplast genome with the transgene were confirmed.
In addition, GFP expression was monitored phenotypically by visualizing green fluorescence under UV light (FIG. 1D).

In order to scale up the biomass of plant leaf material tagged with each GFP, each homoplasmic line was grown in a temperature and humidity controlled automated greenhouse. Fully grown mature leaves were harvested late evening to maximize accumulation of GFP fusion protein driven by light-regulated control sequences. In order to further increase the fusion protein content on a dry weight basis, the frozen leaves were lyophilized at −40 ° C. under vacuum. In addition to the protein concentration effect, lyophilization extended the shelf life of therapeutic proteins expressed in plants for more than a year at room temperature [13].
Thus, in this example, lyophilized and powdered plant cells expressing GFP fusion tag protein were used for oral delivery of mice. Immunoblotting assay of GFP fusion tag protein shows the same size protein in fresh and 4 month old lyophilized leaves (FIG. 1E), stability of fusion protein during lyophilization and extended at room temperature Confirmed saving.
In the immunoblot image, in addition to 39.5 kDa, 29.2 kDa and 28.3 kDa monomers of CTB-GFP, PTD-GFP and DCpep-GFP, respectively, PTD-GFP (58.4 kDa) and DCpep-GFP A dimer of GFP (56.6 kDa) was also detected (FIG. 1E). Homodimerization is one of the physicochemical characteristics of GFP that occurs in solution and in crystals. The contact between the monomers is very stiff due to a wide range of interactions consisting of a hydrophobic side chain core from each monomer and multiple hydrophilic contacts [43].
CTB monomers are also very stable and resistant to heat and denaturing agents due to intersubunit interactions within the pentameric structure mediated by hydrogen bonds, salt bridges and hydrophobic interactions [44]. Can self-assemble into a pentameric structure.
For CTB-GFP, PTD-GFP and DCpep-GFP, the GFP protein concentrations in the powder lyophilized leaf material were 5.6 μg / mg, 24.1 μg / mg and 2.16 μg / mg, respectively (FIG. 1C). . The GFP protein concentration after lyophilization increased by 17.1 times, 12.7 times and 18.8 times for CTB-GFP, PTD-GFP and DCpep-GFP, respectively (FIG. 1F). Removal of water from fresh leaves by lyophilization results in a 90-95% reduction in weight. This effect then appears as a 10-20 fold increase in protein per gram of dry leaf [13, 15, 45].

Uptake of GFP in different tissues after oral delivery of plant cells For oral delivery, lyophilized plant cells (20 mg) were rehydrated in a uniform volume (200 μl) and similar duration. The dispersed plant cells do not change in size because the size of mature plant cells is uniform. As shown in FIG. 2, the biodistribution of GFP does not show a significant change. After oral delivery of lyophilized plant cells expressing GFP (fused with PTD or CTB or DC peptide), systemic GFP levels were higher in PTD-GFP fed animals than any other tag tested (FIG. 2A).
Biodistribution to the liver and lungs was substantially higher than other tissues (skeletal muscle, kidney). GFP levels in these tissues were consistently highest for the PTD fusion protein (Figure 2B). Immunohistochemical studies using GFP-specific antibodies provided further insight into the delivery route.
As shown in FIG. 3A (3C insertion), a sensitive detection method for GFP using Alexa Floor 488-labeled secondary antibody reveals that the PTD tag drives certain GFP uptake by intestinal epithelial cells. did.
No GFP was detected in tissues derived from mice that did not use primary antibodies or were fed non-transformed tobacco cells (FIG. 3B). In order to more easily find the region of the intestine where plant cells are located and where antigen uptake can be observed, the small intestine was rolled before fixation so that the proximal and distal portions were visible on the same slide ( FIG. 3C).
The presence of plant cells that express GFP during the villi of ileum (FIGS. 3C and E) provides the first direct evidence to protect the plant cells from the digestive system. When CTB tags were used, more extensive delivery of GFP to epithelial cells was also seen (FIG. 3D) and this is due to efficient targeting of the GM1 receptor by CTB pentamers. In addition to delivery to epithelial cells, we also found evidence for GFP uptake by M cells with all fusion tags (solid arrows in FIGS. 3C-F).
Again, these findings provide direct evidence for protein uptake in the upper gut after dissolution in the gut.
FIG. 3E shows, among other things, GFP ⁺ plant cells (“ PC "). For DCpep-GFP, no GFP ⁺ epithelial cells were observed. However, an example of colocalization of GFP and M cells was found (FIG. 3F), suggesting that systemic delivery of DCpep-GFP is possible by transport from the intestinal lumen via M cells.
Since blood can carry antigen from the intestine to the liver via the portal vein (gut-liver-axis), all three tags, as expected GFP delivered with showed an accumulation in the liver (FIGS. 3H, J and L).

Purification of GFP fused to different tags To study the uptake of GFP by different cell types, use the PTD-GFP and DC-pep-GFP columns with a toyopearl butyl column and CTB-GFP with a Ni ²⁺ column. Used to purify the GFP fusion protein. To examine purity, densitometry was performed using Western blot and GFP standards (FIG. 4A). The purity of GFP fused to each tag was ˜95% for PTD-GFP, ˜52% for DCpep-GFP, and ˜13% for CTB-GFP.
The variation in the purity level is attributed to the difference in the expression level of each tag reflected in the GFP fusion protein recovered after purification. Since proteins are purified based on hydrophobic interactions, other hydrophobic proteins may be present in the purified fraction.
Purification of CTB-GFP was performed using an affinity Ni ²⁺ column. When the pentameric structure of CTB is formed, a histidine cluster is generated and the imidazole ring of the histidine cluster interacts with Ni ²⁺ [46]. The low purity of CTB-GFP is due to a more stringent wash step, but increasing stringency is associated with higher loss of fusion protein.
Purified GFP fusion proteins in SDS-PAGE and Coomassie stained gels were predicted sizes (29.2 kDa, 28.3 kDa) for PTD-GFP, DCpep-GFP and CTB-GFP, respectively. And 39.5 kDa) showed distinct bands for each fusion protein (FIG. 4B). In the case of PTD-GFP, there were two bands around the expected size, which were 29.2 and 28.3 kDa. The C-terminal tail of GFP (His-Gly-Met-Asp-Glu-Tyr-Lys) is highly cleaved for proteolytic degradation by carboxypeptidases and non-specific proteases including proteinase K and pronase and various isoforms. It has been reported that it is easy to cause by partial proteolytic cleavage and is produced by ion-exchange chromatography, isoelectric focusing, and native gel electrophoresis [47].
To quantify GFP fluorescence, non-denaturing SDS-PAGE was performed, and the fluorescence intensity was strongest in PTD-GFP (lane 1) (FIG. 4C).
In DCpep-GFP (lane 2), a top band with the highest likelihood of showing dimerized GFP was observed [47], three different proteins were seen, which were also observed with PTD-GFP as well. It was much higher intensity.
The CTB-GFP fusion showed a set of larger fluorescent bands (lane 3) starting from 190 kDa corresponding to the pentamerization of CTB fusion GFP and possibly other higher bands indicating multimers. In the same lane, a smaller fragment was observed, slightly lower than the GFP standard, which could be a different fold product, similar to that observed with PTD-GFP and DCpep-GFP.
To assess the proper formation of purified CTB-GFP pentameric structure, a GM1 binding assay was performed using anti-CTB and anti-GFP antibodies. It is well known that the pentameric structure of CTB has a strong binding affinity for the ganglioside GM1 receptor ubiquitous on the surface of mammalian cells [48]. As seen in FIG. 4D, only CTB and CTB-GFP showed binding affinity, indicating the formation of a complex between CTB and GM1.
Moreover, the interaction of GM1-CTB-GFP was reconfirmed using the anti-GFP antibody.
Only CTB-GFP can be detected (FIG. 4E).
To further confirm the pentameric structure of CTB-GFP, purified CTB-GFP was run on a modified Tris-Tricine gel under non-denaturing conditions [36] and using anti-CTB antibody Probed.
The expected pentameric CTB-GFP form was detected at ˜200 kDa along with the 39.5 kDa monomeric form (FIG. 4F).
Due to SDS added in the gel and electrophoresis buffer, the monomers can dissociate from the oligomeric structure during the run. Thus, the CTB-GFP fusion protein formed a pentameric structure and retained the ability to bind to the GM1 receptor, but has no GM1 binding affinity for PTD-GFP or DCpep-GFP fusion proteins.

Uptake of GFP fused with different tags by human immune and non-immune cells Purified GFP fusion protein was incubated with cultured human cells. Blood monocyte-derived mature DCs, T cells (Jurkat cells), B cells (BCBL1), differentiated macrophages and mast cells were cultured for in vitro studies. As examples of non-immune cells, human kidney cells (HEK293T) and human pancreatic epithelial cancer cells (PANC-1) were tested in parallel. Cells (2 × 10 ⁴ ) were incubated with purified CTB, PTD and DC target peptide fusion GFP at 37 ° C. for 1 hour. Incubation with DCpep-GFP, intracellular GFP signal was detected only in DC and not in other cell types, confirming its specificity (FIG. 5A).
PTD-GFP entered kidney cells or pancreatic cells but not any immunoregulatory cells (FIG. 5A).
The PTD sequence is derived from PDX1, which induces insulin expression in pancreatic cells, and exogenous PDX1 can enter the mouse islet cell line (insulinoma cell line) and activate the insulin gene [41]. As expected, a strong GFP signal was observed from PANC-1 cells when incubated with purified PTD-GFP. PTD-GFP was observed in the nucleus of pancreatic duct epithelial cells (FIG. 5B). The cell penetration ability of PTD was also evident in human kidney cell lines (FIG. 5A).
In sharp contrast, GFP signal was detected upon incubation with CTB-GFP in all cell types, consistent with the ubiquitous presence of GM1 receptor (FIG. 5A).
Mouse mast cells from the spinal cord (cell types that play an important role in wound healing, defense against pathogens, and allergic reactions) do not show internalization of GFP delivered by DCpep or PTD, but CTB-fused GFP is efficient (FIG. 5A).
Although only one representative image is presented here, uptake studies were performed in triplicate, and for each cell line, 15-20 images were recorded under a confocal microscope. It was done. CTB-GFP is observed in 70-92% of all cell types examined, and the variation is due to differences in cell density that favor low availability of GFP for their uptake. In the case of PTD-GFP, no uptake was observed in any other cell type except kidney cells and pancreatic cells (0%). DCpep-GFP was not observed in other cell types other than dendritic cells (0%) (Table 1).
This study provides the specificity of delivering protein pharmaceuticals to the serum, immune system or specific organs or tissues, thereby facilitating further advances in this new concept.

Table 1: Uptake efficiency of purified GFP fusion protein in human cell lines Relative delivery efficiency of GFP to human cell lines by three different tags, GFP signal at 100x magnification under confocal microscope Comparison was made by counting the number of cells shown.
A total of 15-20 images were observed for each cell line.
All cell lines were examined in triplicate.

4). Discussion In our previous publication [6-16], plant cell-derived proteins fused with CTB for various applications, including delivery of functional proteins to the circulatory system and protein antigens to the immune system. It was shown that it can be delivered via the intestinal epithelium (as an oral tolerance induction or booster vaccine).
This is possible because CTB pentamer binds to the GM1 receptor on the surface of intestinal epithelial cells and M cells, followed by transcytosis. However, GM1 is widely expressed by many different cell types and it is difficult to target specific cell types. Therefore, this study sought to develop alternative tags that favor either more cell type specific and / or more efficient transmucosal delivery.
As an example of specific delivery to professional antigen-presenting cells, DCpep was selected and its specificity could be confirmed. PTD not only more effectively delivers GFP cargo to the circulation via intestinal epithelial cell invasion (FIGS. 3A and 3C), but completely avoids delivery to immune cells (FIG. 5A). ).
In our opinion, the data presented here for PTD is in stark contrast to the conventional assumption that it is non-specific, a paradigm shift in drug delivery.
Given the recent disagreement in the literature about the mechanism of cell penetration by PTDs (macropinocytosis vs. direct migration) and the effects of PTDs vs. their carrying cargo (49), this goes to this field Is an extremely timely contribution. Our data show that the specific choice of PTD allows the body's immune system to be excluded from protein drug delivery, while being efficiently delivered to other cell types and the circulatory system. This study therefore provides a new approach to increase the specific targeting of therapeutic agents to selected tissue types using preselected PTDs. The array and structural knowledge can be combined to generate a custom designed PTD. We used a defined in vitro system to demonstrate differences in protein transduction between different tags as a function of the target cell type. This approach can also be used to assess immunological outcomes when disease specific antigens and corresponding animal models are used instead of GFP.

Oral delivery of leaf material expressing three GFP-tagged proteins to reveal routes through which the GFP fusion protein passes through the intestinal epithelium and is distributed to the circulation and biodistributed to other organs Later immunostaining studies of small intestine tissue sections were performed.
It was confirmed that CTB can be targeted by M cells to the GM1 receptor on epithelial cells. Our studies have found that PTDs can penetrate non-immune cells and explain why this tag also transfers GFP to epithelial cells.
In contrast, DC peptides are specific ligands for dendritic cells and therefore do not target epithelial cells. However, our data show that uptake by M cells is a mechanism for the entry of DCpep fusions, and thus how systemic delivery of DCpep-GFP via the oral route is possible. explain.
Dendritic cells are also directly targeted by DCpep via their projections between epithelial cells.
However, the amount of intestinal antigen collected by this mechanism is too small to be visualized.

We find that protein antigens released from CTB after transcytosis are taken up by DCs (and to a lesser extent by macrophages) in the intestinal immune system (as widely published). And DCpep-GFP that is translocated by M cells is also expected to be taken up by DC. However, for early studies of interaction with immune cells we utilized a defined in vitro approach that was more sensitive and used cultured human cells.
The CTB fusion delivered GFP to all tissues and cell types tested, including non-immune and immune cells. In our laboratory, it is well established that CTB specifically binds to GM1 ganglioside and that various CTB fusion proteins expressed in chloroplasts also show strong binding affinity with GM1. [6, 8, 9, 13-16]. Once CTB binds to GM1, enriched in membrane lipid rafts of intestinal epithelial cells, the endoplasmic reticulum (trans-Golgi Network) via the trans-Golgi network for cell entry. Go retrograde to endoplasmic reticulum (ER) [50, 51].
Indeed, CTB has been widely used as a probe to quantitatively study GM1 and its cellular and subcellular distribution [52]. CTB was conjugated via strong binding affinity to GM1 and large mucosal regions of human intestine (approximately 1.8-2.7 m ^{2 to} body weight) by using as a transmucosal carrier. It can facilitate the transport of proteins into the circulation [53].
Up to 15,000 CTB molecules can bind to one intestinal epithelial cell at a time [54], and the GM1 receptor is turned over on the cell surface [55]. Furthermore, GM1 ganglioside has also been found in the plasma membrane of many other cell types, particularly abundant in the nervous system and retina [56, 57], and thus CTB fusion proteins in these cells Leading to efficient uptake.
Our strategy for inducing oral tolerance to therapeutic proteins used for replacement therapy of autoantigens and genetic disorders (such as hemophilia and lysosomal storage disorders) is the release of antigen from CTB tag and uptake by DC [ 7, 9, 10, 11, 33], relying in part on efficient targeting of intestinal epithelial cells by CTB, following transmucosal delivery and proteolytic cleavage.
However, DCs and macrophages also directly collect antigens in the intestinal lumen, and M cells can carry intact antigens across the epithelium to DC-rich areas. Our new data show that CTB fusions are efficiently taken up by DCs, macrophages, and other immune cells, and the effectiveness of CTB fusion antigens in plant-based immunoregulatory protocols. Provide further explanation.

Specific targeting of dendritic cells by DCpep, ideal for delivery to the immune system Here are the potential, alternative and more specific peptide sequences for immunotherapy. DCpep specifically targets DC but does not target other immune or non-immune cells. To assess the effects of translation, we mainly used human immune cells to distinguish the target properties of the fusion tag. DCpep delivered only the complete GFP antigen to DC but not to any other APC or immune or non-immune cells. Consistent with this finding, DCpep-GFP was unable to target intestinal epithelial cells in vivo. Systemic delivery is most likely due to uptake by M cells. In the future, depending on the activation signal, immune tolerance and vaccine protocols can be designed based on specific delivery to DC, which has important functions in Treg induction and immune stimulation.

As we know, delivery of antigen to lymph nodes is very important for immunotherapy. However, this aspect is that proteins taken up by DCs in lamina propria or Peyer's patches until DCs migrate to lymph nodes to present antigen to T cells Is fragmented into small peptides and loaded onto MHC molecules, so it cannot be directly demonstrated by the GFP signal.
Consistent with the notion that mesenteric lymph nodes (MLN) are important in response to ingested antigens, we recently increased in different DC subsets of mice that received oral delivery of transplastomic plant cells and Induction of Treg in MLN was demonstrated [10].

PTD is ideal for efficient systemic delivery by the oral route, excluding the immune system CTB fusions effectively target the intestinal immune system and are useful for tolerance induction, but immune complications An alternative strategy to avoid immunocomplications is to minimize interaction with the immune system.
The protein transduction domain of PDX-1 showed a unique selectivity in the transfer of GFP to different cell types. PTD-GFP completely failed to deliver antigen to APCs and lymphocytes, but was able to transfer GFP to non-immune cells (including intestinal epithelial cells in vivo). Since myeloid and lymphoid cells are hematopoietic cells, PDX-1 may not be able to transduce this particular cell line. PDX-1 induces insulin expression during protein transduction via macropinocytosis, a specialized form of endocytosis, distinct from receptor-mediated uptake [59, 60]. .
Since macropinocytosis is also the main mechanism of macromolecular uptake in the kidney, after incubation with purified PTD-GFP, the observation of the GFP signal in HEK293T is the result of induction of endocytosis by PTD. obtain.
The lack of GFP signal in immune cells after incubation with PTD-GFP
i) Lack of cell surface binding, and ii) HIV tat PTDs, which also utilize macropinocytosis mechanisms, convert intact GFP, human DCs and other APCs by means of HIV tat PTDs. In order to deliver easily [61-63], it reflects the failure of protein transduction of these cells, which cannot be explained by enhanced degradation after uptake.
PDX-1 derived PTDs clearly show a clear selectivity of cell transduction, possibly related to the surface properties of the target cell membrane.
Both PTDs enter the cell by macropinocytosis, but their amino acid sequences are very different, which is likely to affect cell surface binding. Lymphocyte infection is an important step in the HIV life cycle, but insulin expression must be tightly controlled and respond to environmental stimuli. [64] This may be partly related to the selectivity of PTD from PDX-1.
At the same time, PTD-GFP excels in vivo in systemic protein delivery that can be used for treatments requiring specific protein levels in the blood, such as published examples of hypertension and hormone or cytokine therapy treatments. [9, 11, 14, 15].
Interestingly, despite significant differences in systemic delivery, CTB-, DCpep- and PTD-tags were all very similar in the liver as evidenced more quantitatively by immunohistochemistry and ELISA. GFP antigen levels were masked.
The association between responses in the gut and liver has long been known and is often referred to as the “gut-liver axis”.
Once taken up by the intestine, the antigen can be transported indirectly to the liver via a migratory DC, a pathway through the mesenteric lymph node. Alternatively, blood can carry the antigen from the intestine to the liver via the portal vein. Given the broad distribution of quantifiable levels of GFP in the liver, the latter explanation seems more likely for our delivery system.
The data suggests that the liver takes up orally delivered antigen to a saturation level that is less dependent on the tag.

Unique advantages of delivering protein bioencapsulated in plant cells There are several advantages to lyophilization of plant cells. Lyophilized powder leaves can be stored at room temperature for several years, eliminating the expensive refrigeration and transport required for injectable protein drugs [13,65]. Also, the concentration effect of the therapeutic protein increases to promote a 10-20 fold reduction in the size of the capsule containing the lyophilized plant cells.
Lyophilization techniques are widely used by the pharmaceutical industry for the storage of protein drugs, including the storage of blood clotting factors. Thus, the lyophilization process does not denature the protein. In fact, we have repeatedly shown that lyophilization preserves proper folding and disulfide bonds. (11, 13-16, 66). Upon oral delivery, lyophilized plant cells reach the intestine, and digestion of the plant cell wall releases bioencapsulated proteins by intestinal microorganisms. The released proteins and plant cell walls can then be degraded by intestinal microorganisms. However, intestinal microorganisms can be enriched by anaerobic bacteria that release more enzymes to degrade plant cell walls than proteolysis. Bacteria that inhabit the human intestine can actually evolve to utilize complex carbohydrates in plant cell walls and can utilize almost any plant glycan [4,5]. Our previously published work has identified the enzymes necessary to break down plant cell walls [67, 68].
The delivery of some functional proteins indicates that they are released in the proper amount of protein pharmaceuticals that are protected in the intestinal lumen or that can tolerate intestinal proteases.

The DCpep-GFP content was the lowest of the three fusion proteins, 2.16 μg / mg, which was found to be 10 times lower than PTD-GFP. In general, chloroplast expression of foreign proteins can reach very high levels of the entire leaf protein, up to 70%, due to the high copy number of the chloroplast genome [7]. However, expression levels vary based on protein, N-terminal fusion, cleavage and stability in proteolysis. In this study, all chimeric genes were driven by the psbA promoter and psbA 5′UTR and stabilized by the psbA 3′UTR.
Since the GFP sequence is also the same among all three constructs, the contributing factor affecting the difference in expression levels may be attributed to the N-terminal sequence of the fusion construct.
In contrast to PTD and CTB tags, DCpep was fused to the C-terminus of GFP. Thus, one possible explanation for the lower level of GFP expression of the DCpep fusion is insufficient N-terminal protection.

Conclusion As explained above, the bioavailability of oral delivery of protein drugs expressed in genetically modified plant cells currently induces tolerance to autoimmune diseases [7], or Eliminate protein drug toxicity by injection [8-10], or treat diabetes [12,13], hypertension [14], protection against retinopathy [15], or plaque removal in Alzheimer's brain [16] Therefore, delivering functional blood proteins is currently a hot topic as a new concept. As seen in diabetes studies where oral delivery was as effective as injectable delivery to lower blood glucose using insulin or exendin-4, these novel approaches are In addition to significantly reducing health care costs, it should improve patient compliance [12,13].
This study allowed the use of different fusion tags to deliver to immune regulatory or non-immune cells or directly to serum without interfering with the immune system. This opens up the possibility of low-cost oral delivery to regulate metabolic pathways, enhance or suppress immune or functional proteins. The cost of protein drugs is currently over 75% of GDP in countries and is out of reach. This is due to their very expensive fermenter production, purification, refrigerated storage / transport, short shelf life, and sterile delivery methods.
Using green fluorescent protein (GFP) as a model, we have shown in this study that plant cells protect GFP from the digestive system and release it into the intestinal lumen where it is absorbed by epithelial cells. Demonstrate. Based on delivery tags fused to GFP, they reach the circulatory system, immune or non-immune cells, specific organs or tissues.
Such oral delivery of low cost protein pharmaceuticals increases patient compliance and dramatically reduces costs by eliminating the very expensive processes / injections currently in use.

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Example II
Evaluation of uptake of GFP fused with different tags: CTB-GFP, PTD-GFP, PG1-GFP and RC101-GFP in additional human cell lines
In order to expand the types of cells that can be successfully targeted using the protein therapeutics described herein, we evaluated using both human somatic and stem cell lines. Six cell lines were selected with the goal of achieving targeted delivery of therapeutic proteins to benign and malignant tissues. These strains included normal and cancer cell lines from human dentistry, head and neck and soft tissues. Three human stem cell lines were selected to develop functional protein-based therapies for developmental disorders and immature / stem cell tumors.

Figures 6A and 6B show the study results obtained with different cell types and tags. These human somatic cells include pancreatic cancer cells (PANC-1); human pancreatic ductal epithelial cells (HPDE); human head and neck squamous cell carcinoma cells (SCC-1); retinal pigment epithelial cells (RPE); adult gingival keratinocytes (AGK); including osteoblasts (OBC). Certain types of human stem cells were also evaluated. These include human periodontal ligament stem cells (HPDLSC); Maxillar mesenchymal stem cells (MMSC) and Gingiva-derived mesenchymal stromal cells (GMSC).
CTB, PTD and DC-peptide fusion tags are described in Example I. Protegrin (PG) and retrocycline (RC) are antibacterial peptides and are described in detail in Lee et al., Plant Biotechnology Journal 9: 100-115 (2011).
Protegrin 1: Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Arg-Arg-Phe-Cys-Val-Cys-Val-Gly-Arg (SEQ ID NO: 5),
Protegrin 2: Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Arg-Arg-Phe-Cys-Val-Cys-Val (SEQ ID NO: 6)
Protegrin 3 replaces arginine at position 4 with glycine and also has one less positive charge. Protegrin 4 replaces valine at position 14 with phenylalanine and differs in sequence at the β-turn. This difference makes protegrin 4 less polar and less positively charged than the others. Protegrin 5 replaces arginine with proline and has one less positive charge.
Additional retrocycline variants are provided in PCT / US2002 / 012353, incorporated herein by reference.

Results PANC-1 cells:
In human pancreatic cancer cells, CTB-GFP shows moderate accumulation in the cell membrane and a scattered foci is seen in the cytoplasm. PTD-GFP is localized in the cell membrane as well, but the signal is stronger in the cytoplasm than that of CTB-GFP. PG1-GFP shows a very strong accumulation in the cell membrane, but only a weak scattered signal in the cytoplasm. RC101-GFP exhibits the strongest GFP levels in both the cell membrane and the cytoplasm.

HPDE cells:
In human pancreatic ductal epithelial cells, CTB-fused GFP exhibits a relatively strong, high-density signal in the cell membrane and cytoplasm. PTD-GFP does not give a clear signal at the cell membrane, but has a high density and moderate signal in the cytoplasm and nucleus. PG1-GFP shows a strong accumulation in the cell membrane, but only shows a scattered focus in the cytoplasm. RC101-GFP showed punctate GFP localization only at the cell membrane.

HPDLSC cells:
In human periodontal ligament stem cells, CTB-GFP shows a relatively strong signal only in the cell membrane. PTD-GFP shows very weak accumulation at the cell membrane, but has a scattered, moderately bright GFP signal in the cytoplasm. PG1-GFP shows strong localization in both cell membrane and cytoplasm. RC101-GFP cannot localize to both the cell membrane and cytoplasm of these cells.

MMSC cells:
In maxillar mesenchymal stem cells, CTB-GFP shows a relatively strong dense localization and scattered spots in the cytoplasm at the cell membrane. PTD-GFP does not accumulate in the cell membrane but is cytoplasmic and exhibits a moderately bright focus. PG1-GFP shows a very strong GFP signal at the cell membrane and a strong signal scattered in the cytoplasm. RC101-GFP does not appear to be localized anywhere in these cells.

SSC-1 cells:
In human head and neck squamous carcinoma cells, CTB-GFP exhibits a weak GFP signal in both the plasma membrane and the cytoplasm. PTD-GFP has a relatively strong localization in the cell membrane and a strong signal in the cytoplasm. PG1-GFP shows moderate accumulation in the cell membrane and shows a signal scattered in the cytoplasm. RC101-GFP exhibits weak GFP localization in the cell membrane and a small number of scattered foci in the cytoplasm.

RPE cells:
In retinal pigment epithelial cells, CTB-GFP is localized in the cell membrane (as opposed to punctate) and exhibits a relatively strong GFP focus in the cytoplasm. PTD-GFP exhibits a relatively strong accumulation in the cell membrane and a strong clustering in the cytoplasm. PG1-GFP shows the strongest signal of any peptide in these cells, both at the cell membrane and cytoplasmic focus.

GMSC cells:
In gingiva-derived mesenchymal stromal cells, CTB-GFP is localized in both the cell membrane and the cytoplasm. PTD-GFP exhibits a relatively strong GFP accumulation in the cell membrane and a strong clustered focus in the cytoplasm. PG1-GFP shows a moderate accumulation of GFP at the cell membrane and a strong clustered signal in the cytoplasm.

AGK cells:
In adult gingival keratinocytes (adult gingival keratinocytes), CTB-GFP is cytoplasmic and exhibits many relatively strong, high-density signals. PTD-GFP also shows a relatively strong signal in the cytoplasm, but there is relatively little focus. PG1-GFP shows a much stronger accumulation in the cytoplasm.

OBC cells:
In osteoblasts, CTB-GFP shows strong, punctate localization in the cytoplasm. PTD-GFP is also strongly localized in the cytoplasm. Only PG1-GFP exhibits high density and continuous localized GFP in the cell membrane.

Discussion Upon incubation with CTB-GFP, the GFP signal is detected in all cell lines, and the expression pattern in these cells is similar, and the ubiquitous presence of the GM1 receptor on the membrane of these cells Was consistent. The protein transduction domain (PTD) also effectively facilitated the delivery of GFP to these cell lines, and also delivered GFP even to the nucleus of HPDE cells. These results confirm that PTD can deliver foreign proteins directly without membrane receptors.
Protegrin 1 (PG1) fusion GFP shows strong localization in the cell membrane and cytoplasm of most cell lines. There are three expression patterns:
1. Strong GFP signal at both cell membrane and cytoplasm (MMSC, RPE) indicating that PG-1 can penetrate the cell membrane.
2. Cytoplasmic signals (PANC-1, HPDE and OBC) with strong GFP signal at the cell membrane but weak or absent indicate that PG-1 is unable to penetrate the membrane of these cells.
3. There is a weak GFP signal in the cell membrane, but a strong signal in the cytoplasm (SCC-1, HPDLSC, GMSC and AGK).
This pattern indicates that PG1 penetrates the membranes of these cells most easily and efficiently and efficiently transports protein drugs to the cytoplasm. These results indicate that the efficiency of PG1-GFP penetration varies with cell type.

Protein Drug Delivery Targets Using Fusion Tags Fusion between CTB and therapeutic protein is the effect of therapeutic protein even to induce oral tolerance, delivery to serum, or even penetrate the blood-brain or retinal barrier Facilitating oral delivery. A foreign protein can be delivered to living cells by fusing it with a protein transduction domain (PTD), which can enter the cell membrane independently of a specific receptor. PTD delivers biologically active proteins in cultured mammalian cells and animal models in vivo and in vitro, giving PTD fusion proteins great potential for therapeutic drug delivery. Although antimicrobial peptides are known to kill microorganisms, human cell specific targeting is a new and unexpected observation. Here we have antimicrobial peptides that can perform this dual function, making antimicrobial peptides ideal candidates for use as peptide antibiotics, as well as in cell-specific ways, This is for effective delivery of other protein drugs. Some examples of additional protein drugs are shown below.
The amino acid sequence of the GFP reporter protein comprises the therapeutic proteins listed in Table 4, including anti-inflammatory functional drugs, pancreatitis, treatment of periodontitis and periodontitis, new treatment of pancreatic cancer and head and neck squamous cell carcinoma It can be replaced with the encoding nucleic acid.
In the field of periodontal regeneration, several protein-based therapeutics have been shown to be effective. Fusion of such proteins with the tags of the present invention, as well as administration of plant cells or plant parts that express such fusion proteins, provides efficacy for the treatment of various dental disorders.

Table 3 provides a list of cells that can be selectively targeted by fusion with different tags of the present invention.

  In addition, the approaches described herein can be used to deliver pharmaceuticals useful for the medical indications described.

Replacing defective proteins for the recognized disorders
Endocrine disorder (hormone deficiency)
Insulin (analog with faster action, longer duration)
Growth hormone (somatotropin)
Insulin-like growth factor

Hemostasis and thrombosis (post translational changes) (Hemostasis and thrombosis (post translational changes))
Blood coagulation factors (VII, VIII, IX)
Antithrombin (surgery)
Protein C (venous thrombosis)
Tissue plasminogen activator (embolism, stroke)

Replacing defective enzymes
Enzyme-deficient β-glucocerebrosidase α-glucosidase

Lung / gastrointestinal disorder proteinase inhibitor (antitrypsin deficiency)
Lactase lipase amylase protease

The therapeutic fusion proteins of the present invention can also be used to extend existing pathways, such as those involved in hematopoiesis and fertility. Thus, creating a fusion protein comprising erythropoietin (anemia, chemotherapy), granulocyte colony stimulating factor, granulocyte macrophage stimulating factor, adenosine deaminase, human serum albumin (nephrotic syndrome), immunoglobulin, follicle stimulating hormone and chorionic gonadotropin be able to. Different interferons such as interferon alpha (hepatitis C, anti-virus B) and interferon beta (multiple sclerosis) can be manufactured and administered as described herein.
Hormones such as parathyroid hormone and exenatide can also be produced as selectively targeted fusion proteins in accordance with the present invention.

Bone morphogenetic proteins for the treatment of spinal fusions, gonatropin-releasing hormone for adolescent use, keratinocyte growth factor for oral mucositis associated with chemotherapy, and platelet-derived growth factor for wound healing Can be used.
Other protein-based drugs can also be fused to the fusion peptides of the invention to improve targeted delivery.

These include, but are not limited to, botulinum toxin for dystonia and cosmetic applications, collagenase for the treatment of severe burns, DNase for cystic fibrosis, papain for burns and ulcers, asparaginase for leukemia, coronary arteries Hirudin for plastic surgery and DVT or streptokinase for embolism are included.
Additional effective agents can also be used in the form of fusion proteins of the invention and are listed in Table 4.

  For details on this table and other protein pharmaceuticals, see Walsh, G (2014), Biopharmaceutical benchmarks 2014, Nat. Biotechnol. 32,992-1000.

  While some of the preferred embodiments of the present invention have been described and specifically implemented, the present invention is not intended to be limited to such embodiments. Various modifications can be made without departing from the scope and spirit of the invention as set forth in the appended claims.

Claims (19)

A method for targeted delivery of a therapeutic protein to a tissue or cell of a subject's interest for treating a disease or disorder in the subject comprising the steps of:
a) administering an effective amount of plant cells or remnants thereof comprising a nucleic acid expressing a plastid encoding the therapeutic protein operably linked to a fusion peptide sequence. Wherein expression of the nucleic acid causes production of a therapeutic fusion protein in the plastid; and
b) caused in vivo by selective penetration of the therapeutic fusion protein into the tissue or cell of interest, substantially excluding non-target cells (to the substitutive of non target cells in vivo) Ameliorate or alleviate symptoms associated with the disease or disorder,
Including methods. The method of claim 1, wherein the plant is selected from the group consisting of lettuce, tobacco, spinach, kale, cabbage, eggplant, carrot and tomato. The method of claim 1, wherein the peptide is a PTD peptide. The method of claim 1, wherein the peptide is a DC peptide. The method of claim 1, wherein the peptide is an antimicrobial peptide. 6. The method of claim 5, wherein the peptide is a PG1 peptide or RC101 peptide. The method of claim 1, wherein the peptide is a CTB peptide. The method of claim 1, wherein the cell is an immune cell. The method of claim 1, wherein the cell is a somatic cell. The method of claim 1, wherein the cell is a dendritic cell. 11. The method of any one of claims 1-10, wherein the therapeutic fusion protein is for the treatment of an endocrine disorder and is selected from the group consisting of insulin, somatotropin, and insulin-like growth factor. The therapeutic fusion protein is for maintenance of hemostasis or prevention of thrombosis, and the protein is selected from the group consisting of blood clotting factor, antithrombin, protein C and tissue plasminogen activator The method of any one of Claims 1-10. The subject has an enzyme deficiency and the therapeutic fusion protein is selected from the group consisting of β-glucocerebrosidase, α-glucosidase, lactase, lipase, amylase, protease and proteinase inhibitor. The method of any one of 10-10. The therapeutic fusion protein enhances existing therapeutic protocols and erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, follicle stimulating hormone, chorionic gonadotropin, alpha interferon, interferon B, PDGF, keratinocyte growth 11. A method according to any one of claims 1 to 10, selected from the group consisting of factors and bone morphogenetic proteins. 11. The method of any one of claims 1-10, wherein the therapeutic protein is provided in Table 4. A plant or plant cell comprising the therapeutic fusion protein according to any one of claims 1 to 15. 17. Plant cells according to claim 16, which are lyophilized and optionally in powder form. 17. A plant cell according to claim 16, which is encapsulated. 17. The plant cell of claim 16, wherein the therapeutic fusion protein is stable at ambient temperature.